Cytotoxic Activity and Lymphocyte Subtypes in Mice Selected for Maximal and Minimal Inflammatory Response after Transplantation of B16F10 and S91 Melanoma Cells.

AIRmax and AIRmin mice strains were selected according to the intensity of their acute inflammatory responsiveness. Previous studies have shown that AIR mice differ in their resistance to chemically induced skin tumors and in the development of melanoma metastases, in addition to differences in neutrophil and NK cells activity. In the present work, we aimed to evaluate whether the difference of susceptibility to murine melanoma is associated with NK cytotoxic activity against Yac.1 cells and lymphocyte subsets. Mice were subcutaneously inoculated with B16F10 or S91 melanoma cells. After 7, 14, or 30 days, the animals were euthanized to analyze the number of lymphocyte subsets, cytotoxic activity, and number of cytokine-producing spleen cells. AIRmax mice presented a higher number of CD4+/CD25+ cells than that of AIRmin mice following inoculation of B16F10 cells, whereas inoculation of S91 cells reduced CD4+/CD25+ and increased TCD8+ cell subsets in the AIRmax mice. AIRmax mice had a higher number of interleukin (IL)-10- and IL-12-producing cells and a lower number of interferon-γ-producing cells than those of AIRmin mice at 30 days. The cytotoxic activity of nonadherent spleen cells was similar in both the AIR strains. These results suggest that melanoma cells can induce different responses in AIR mice, possibly owing to alterations in regulatory mechanisms, such as the action of CD4+/CD25+ regulatory T cells and IL-10, in AIRmax mice.

Brazilian green propolis: A novel tool to improve the cytotoxic and immunomodulatory action of docetaxel on MCF-7 breast cancer cells and on women monocyte.

Docetaxel (DTX) is used against breast cancer despite its side effects such as toxicity and immunosuppression. Here we investigated the cytotoxic and immunomodulatory effects of the ethanol solution extract of propolis (EEP) in combination with DTX on MCF-7 breast cancer cells and on women's monocyte. The cytotoxic potential of EEP + DTX was assessed by MTT assay and the type of tumor cell death was evaluated by flow cytometry. The effects of EEP + DTX on the migration and invasion of MCF-7 cells were analyzed. Cytokine production by monocytes was assessed by ELISA and the expression of cell surface markers was evaluated by flow cytometry. We also assessed the fungicidal activity of monocytes against Candida albicans and the generation of reactive oxygen species (ROS). Finally, the impact of the supernatants of treated monocytes in the viability, migration, and invasiveness of tumor cells was assessed. EEP enhanced the cytotoxicity of DTX alone against MCF-7 cells by inducing necrosis and inhibiting their migratory ability. EEP + DTX exerted no cytotoxic effects on monocytes and stimulated HLA-DR expression, TNF-α, and IL-6 production, exerted an immunorestorative action in the fungicidal activity, and reduced the oxidative stress. Our findings have practical implications and reveal new insights for complementary medicine.

Propolis increases Foxp3 expression and lymphocyte proliferation in HIV-infected people: A randomized, double blind, parallel-group and placebo-controlled study.

HIV infection and the prolonged use of antiretroviral therapy (ART) contribute to persistent inflammation and immune deregulation in people living with HIV/AIDS (PLWHA). Propolis is a bee product with plenty of biological properties, including immunomodulatory and anti-inflammatory action. This work aimed to evaluate possible changes in the immune/inflammatory response in PLWHA under ART after propolis intake. Asymptomatic PLWHA were double-blindly randomized into parallel groups receiving propolis (500 mg/day, n = 20) for 3 months or placebo (n = 20). Plasma cytokines (TNF-α, IL-2, IL-4, IL-6, IL-10 and IL17) were evaluated by cytometric bead array; cytokine production by PBMC (IFN-γ, IL-5, IL-17, IL-10, IL-1ß, IL-18, and IL-33) was assessed by ELISA; gene expression (T-bet, GATA-3, RORγt and Foxp3) was determined by RT-qPCR, and cell proliferation was analysed by flow cytometry using CFSE staining. The average of gender, age, CD4+/CD8+ T cell count, time of diagnosis and treatment were similar in both groups. No differences were observed in cytokine levels nor in inflammasome activation. However, Pearson's correlation showed that IL-10 was directly correlated to CD4+ T cell count and inversely to IFN-γ after treatment with propolis. Foxp3 expression and lymphocyte proliferation increased in the propolis group. Data suggested that daily propolis consumption may improve the immune response and decrease the inflammatory status in asymptomatic PLWHA under ART.

Transcriptional analysis of THP-1 cells infected with Leishmania infantum indicates no activation of the inflammasome platform.

Leishmaniasis is caused by intracellular parasites transmitted to vertebrates by sandfly bites. Clinical manifestations include cutaneous, mucosal or visceral involvement depending upon the host immune response and the parasite species. To assure their survival inside macrophages, these parasites developed a plethora of highly successful strategies to manipulate various immune system pathways. Considering that inflammasome activation is critical for the establishment of a protective immune response in many parasite infections, in this study we determined the transcriptome of THP-1 cells after infection with L. infantum, with a particular focus on the inflammasome components. To this end, the human cell line THP-1, previously differentiated into macrophages by PMA treatment, was infected with L. infantum promastigotes. Differentiated THP-1 cells were also stimulated with LPS to be used as a comparative parameter. The gene expression signature was determined 8 hours after by RNA-seq technique. Infected or uninfected THP-1 cells were stimulated with nigericin (NIG) to measure active caspase-1 and TNF-α, IL-6 and IL-1ß levels in culture supernatants after 8, 24 and 48 hours. L. infantum triggered a gene expression pattern more similar to non-infected THP-1 cells and very distinct from LPS-stimulated cells. Some of the most up-regulated genes in L. infantum-infected cells were CDC20, CSF1, RPS6KA1, CD36, DUSP2, DUSP5, DUSP7 and TNFAIP3. Some up-regulated GO terms in infected cells included cell coagulation, regulation of MAPK cascade, response to peptide hormone stimulus, negative regulation of transcription from RNA polymerase II promoter and nerve growth factor receptor signaling pathway. Infection was not able to induce the expression of genes associated with the inflammasome signaling pathway. This finding was confirmed by the absence of caspase-1 activation and IL-1ß production after 8, 24 and 48 hours of infection. Our results indicate that L. infantum was unable to activate the inflammasomes during the initial interaction with THP-1 cells.

The chemical composition and events related to the cytotoxic effects of propolis on osteosarcoma cells: A comparative assessment of Colombian samples.

Osteosarcoma (OSA) is a type of bone cancer showing an aggressive biological behavior with metastatic progression. Because propolis potential for the development of new antitumoral drugs has been indicated, we evaluated the chemical composition of Colombian propolis samples and the mechanisms involved in their cytotoxic effects on OSA cells. The chemical composition was analyzed by GC-MS and the DPPH free radical scavenging activity was measured. Cluster and principal components analysis were used to establish an association with their inhibitory concentration 50% (IC50 ). Cell viability was analyzed by MTT assay; apoptosis was determined by flow cytometry; mitochondrial membrane permeability and reactive oxygen species were evaluated by rhodamine 123 and DCFH-DA. Transwell assay was used to evaluate the invasiveness of propolis-treated cells. Samples were grouped: Cluster 1 contained diterpenes and benzophenones and showed the highest antiradical activity; Cluster 2 was characterized by triterpenes, fatty acid, and diterpenes. Usm contained diterpenes and triterpenes different of the other samples and Sil contained triterpenes and flavonoids. Apoptosis, mitochondrial membrane alteration, and suppression of cell invasion were the main mechanisms involved in the inhibition of OSA cells in vitro, suggesting the potential of Colombian propolis to discover new antitumor drugs.

Sonic hedgehog drives layered double hydroxides-induced acute inflammatory landscape.

Although layered double hydroxides (LDH) have been listed as promising nanomaterials in human healthcare, very little has been achieved on osteoblast inflammatory signaling. Thus, osteoblasts were challenged with two LDHs (Mg2Al-Cl and Zn2Al-Cl, at 0.002 mg/mL) up to 24 h, establishing an acute inflammatory mechanism, as well as identifying whether Sonic hedgehog (Shh) signaling has an influence. Functional experiments were performed by previously treating (2 h) semiconfluent osteoblast cultures with cyclopamine molecule (cyc), a widely used Shh inhibitor. Considering inflammasome complex, the asc1 gene was significantly up-expressed in response to Zn2Al-Cl - LDHs, as well as the nrlp3 gene. By treating the osteoblast with cyc, the asc1 gene presented an even higher profile. Our results found a down-modulation of major pro-inflammatory cytokines-related genes, when tnfα and il1ß were significantly down-modulated in response to LDHs. Conversely, anti-inflammatory cytokines were up-modulated considering the same experimental procedures. Except the il6, the other il13, il10, and tgfß genes were up modulated. Additionally, Shh signaling seems to modulate this repertory as both the il13 and il10 genes were significantly up-modulated when the Shh signaling was inhibited. Altogether, our results reveal for the first time the exigency of Shh-dependent anti-inflammatory signals in LDH-induced osteoblast responses.

A new chemotherapeutic approach using doxorubicin simultaneously with geopropolis favoring monocyte functions.

AIMS: Chemotherapy has been widely used to treat cancer although it may affect non-target cells involved in the immune response. This work aimed at elucidating whether the chemotherapeutic agent doxorubicin in combination with geopropolis produced by Melipona fasciculata Smith could affect nontumor immune cells, evaluating their immunomodulatory effects on human monocytes. MAIN METHODS: Cell viability, expression of cell markers (HLA-DR, TLR-2, TLR-4, C80 and CD40), cytokine production (TNF-α, IL-1ß, IL-6 and IL-10), intracellular pathways (NF-κB and autophagy), the microbicidal activity of monocytes and hydrogen peroxide (H2O2) production were analyzed. KEY FINDINGS: Data showed that doxorubicin + geopropolis diminished IL-6 secretion, stimulated TNF-α and IL-10 production, TLR-4 and CD80 expression, NF-κB and autophagy pathway, as well as the bactericidal activity. SIGNIFICANCE: Our findings revealed a new chemotherapeutic approach using doxorubicin simultaneously with geopropolis without affecting human monocytes viability and exerting immunomodulatory effects, favoring cell functions. While doxorubicin altered some immunological parameters, the addition of geopropolis compensated some changes.

Toll-Like Receptor-2 and -4 Expression by Maternal Neutrophils in Preterm Labor.

BACKGROUND AND AIMS: The inflammatory response in preterm parturition is regulated by the innate immune system. Toll-like receptors (TLR)-2 and TLR-4 are innate immune receptors that recognize the microorganisms most frequently involved in amniotic cavity infections, which are associated with activating the inflammatory response at the maternal-fetal interface during preterm labor. This study aimed to evaluate the expression of TLR-2 and TLR-4 in maternal neutrophils in preterm labor. METHODS: A cross-sectional study was conducted in the Obstetrics Care Unit of Botucatu Medical School, UNESP, Brazil. The preterm group was composed of 20 pregnant women who presented preterm labor and preterm delivery. The control group was composed of 20 nonlaboring pregnant women matched to the preterm group by gestational age. Neutrophils were isolated from peripheral blood and TLR expressions were performed by real-time polymerase chain reaction and flow cytometry. RESULTS: Gene expressions of TLR-2 and TLR-4 in neutrophils from the preterm group were statistically higher than expressions in neutrophils from the matched control group. The percentage of TLR-4+ neutrophils was higher in the preterm group than the matched control group, while the percentage of TLR-2+ neutrophils did not differ between groups. CONCLUSION: TLR-4 expression in maternal neutrophils is associated with spontaneous preterm labor.

A unique heterologous fibrin sealant (HFS) as a candidate biological scaffold for mesenchymal stem cells in osteoporotic rats.

BACKGROUND: The injection of mesenchymal stem cells (MSCs) directly into the bone of osteoporotic (OP) patients for rapid recovery has been studied worldwide. Scaffolds associated with MSCs are used to maintain and avoid cell loss after application. A unique heterologous fibrin sealant (HFS) derived from snake venom was evaluated for the cytotoxicity of its main components and as a three-dimensional biological scaffold for MSCs to repair a critical femur defect in osteoporotic rats. METHODS: The cytotoxicity of HFS was assessed using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay and transmission electron microscopy. The cells were cultured, characterized by flow cytometry and differentiated into the osteogenic lineage. Two-month-old rats underwent ovariectomy to induce OP. After 3 months, a 5 mm critical bone defect was made in the distal end of the rat femurs and filled with HFS; HFS + MSCs; and HFS + MSCs D (differentiated into the osteogenic lineage) to evaluate the effects. An injury control group (injury and no treatment) and blank control group (no injury and no treatment) were also included. The animals were observed at days 14 and 28 by microtomographic (micro-CT) analyses, histologic and biochemical analysis, as well as scanning electron microscopy. RESULTS: The results revealed that one of the compounds of HFS, the thrombin-like enzyme extracted from snake venom, had no cytotoxic effects on the MSCs. OP was successfully induced, as demonstrated by the significant differences in the levels of 17ß-estradiol, Micro-CT analyses and alkaline phosphatase between the ovariectomized (OVX) and non-ovariectomized (NOVX) groups. The histological data revealed that at 14 days after surgery in both the OVX and NOVX animals, the HFS + CTMs and HFS + CTMsD showed a higher formation of bone cells at the site in relation to the control group (without treatment). Collagen formation was evidenced through bone neoformation in all treated and control groups. No morphological differences in the femurs of the NOVX and OVX animals were observed after the surgical procedure. Scanning electron microscopy (SEM) confirmed the histological analysis. CONCLUSIONS: The new HFS composed of two non-toxic components for MSCs showed capacity to promote the recovery of the bone lesions in OVX and NOVX animals at 14 days after surgery. In addition, the HFS enabled the differentiation of MSCs into MSCs D in the group treated with HFS + MSCs. Using the MSCs and/or MSCs D together with this biopharmaceutical could potentially enable significant advances in the treatment of osteoporotic fractures. Future clinical trials will be necessary to confirm these results.

The involvement of TLR2 and TLR4 in cytokine and nitric oxide production in visceral leishmaniasis patients before and after treatment with anti-leishmanial drugs.

Toll-like receptors (TLRs) have significant involvement in Leishmania infection, although little is known about the relationship between these receptors, cytokines and nitric oxide (NO) in patients with visceral leishmaniasis (VL) before or after treatment with anti-leishmanial drugs. The goal of this study was to evaluate the expression of TLR2 and TLR4 in CD3+ and CD14+ cells and the production of TNF-α, IFN-γ, IL-17, IL-10, TGF-ß and NO in peripheral blood mononuclear cells (PBMCs) from VL patients pre- and post-treatment with anti-leishmanial drugs. In addition, we investigated whether these receptors were involved in the production of these cytokines and NO. In the active VL patients, increased TLR2 and TLR4 expression in lymphocytes and monocytes, increased production of TNF-α, IL-10 and TGF-ß and decreased production of IFN-γ, IL-17 and NO were observed. After treatment, TLR2 and TLR4 were still expressed in lymphocytes and monocytes, the TNF-α and IL-10 levels were lower, the production of IFN-γ, IL-17 and NO was higher, and the TGF-ß level remained high. Before treatment, the production of TNF-α and NO was associated with TLR2 and TLR4 expression, while IL-10 production was only associated with TLR2 expression. After treatment, both receptors were associated with the production of TNF-α, IFN-γ, IL-10 and NO, while the production of IL-17 was associated only with TLR4 expression. The results presented in this study suggest that both TLR2 and TLR4 participate in the modulation of cytokine and NO production in VL patients, contributing to the pathogenesis of VL prior to treatment and the protective immune response after treatment.

The immunomodulatory effect of propolis on receptors expression, cytokine production and fungicidal activity of human monocytes.

OBJECTIVES: Propolis is a beehive product and its immunomodulatory action has been documented; however, little is known concerning its mechanisms of action on human cells. Propolis influence on the initial events of the immune response was assessed, evaluating cell markers, cytokine production and the fungicidal activity of human monocytes. METHODS: Toll-like receptor (TLR)-2, TLR-4, human leukocyte antigen-DR and cluster of differentiation (CD)80 expression by human monocytes was assessed using a FACSCalibur flow cytometer, cytokine production (tumour necrosis factor (TNF)-α and interleukin (IL)-10) was determined by ELISA and the candidacidal activity was investigated after monocytes incubation with propolis and challenged with Candida albicans. The role of TLR-2 and TLR-4 on propolis action was assessed as well. KEY FINDINGS: Propolis upregulated TLR-4 and CD80 expression and affected TNF-α and IL-10 production, depending on concentration. Propolis also increased the fungicidal activity of monocytes. Cytokine production was decreased by blocking TLR-4, whereas the fungicidal activity was affected by blocking TLR-2. CONCLUSIONS: Propolis exerted an immunomodulatory action on cell receptors, cytokine production and fungicidal activity of human monocytes without affecting cell viability and depending on concentration. TLR-2 and TLR-4 may be involved in its mechanism of action.

Bone marrow mesenchymal stem cells stimulated by bFGF up-regulated protein expression in comparison with periodontal fibroblasts in vitro.

OBJECTIVE: The aim of this study was to evaluate, in vitro, the role of bFGF in the proliferation and expression of collagen type I and fibronectin of dog bone marrow mesenchymal stem cells (dBMMSCs) in comparison with the expression of the same proteins in dog periodontal fibroblasts (dPLFs). DESIGN: dBMMSCs from the iliac crest were cultivated in Dulbecco's Modified Eagle's Medium (DMEM). Flow cytometry analysis (FCA) was used to characterize dBMMSC. Cells were stimulated with bFGF (1, 5 and 10 ng/mL) after 24 and 48 h. Real time RT-PCR was performed to verify collagen type I and fibronectin expressions. MTT assay was used to confirm cellular proliferation. Statistical analyses were performed (ANOVA and Kruskal-Wallis tests; p<0.05). RESULTS: FCA showed 55.98% of CD34+ and 32.67% of CD90+ after bone marrow aspiration; 3.33% of CD34+ and 33.0% of CD90+ before P1. After P2, 10.54% of dBMMSCs expressed CD90, whereas after P3, this number decreased to 1.58%. dPLFs presented 4.04% of CD90+ and 1.05% of CD34+ after P3. MTT evaluation showed increase in dBMSC proliferation with 5 ng/mL bFGF-stimulus after 24-h. Both collagen I and fibronectin expression were very similar between the two cells groups after 24-h stimulation with 1 ng/mL bFGF concentration. Fibronectin and collagen I expressions were higher after 24-h stimulation with 5 ng/mL bFGF. CONCLUSION: dBMMSCs (1 ng/mL-bFGF stimulus after 24 h) are very similar to dPLFs as regards morphological and immunostaining characteristics, and collagen and/or fibronectin production. The dBMMSCs presented the highest protein expression rates with 5 ng/mL-bFGF stimulus after 24-h.

Comparison of inflammatory cytokine profiles in plasma of patients undergoing otorhinological surgery with propofol or isoflurane anesthesia.

OBJECTIVE AND DESIGN: The effects of anesthetics on cytokine release in patients without comorbidities who undergo minor surgery are not well defined. We compared inflammatory cytokine profiles in adult patients undergoing minimally invasive surgery who received isoflurane or propofol anesthesia. METHODS: Thirty-four patients without comorbidities undergoing minor surgery were randomly assigned to receive an inhaled anesthetic (isoflurane; n = 16) or an intravenous anesthetic (propofol; n = 18). Blood samples were drawn before premedication and anesthesia (T1), 120 min after anesthesia induction (T2), and on the first post-operative day (T3). Plasma concentrations of interleukins (IL-) 1ß, 6, 8, 10 and 12 and tumor necrosis factor (TNF)-α were measured using flow cytometry. RESULTS: The pro-inflammatory cytokine IL-6 was increased in the isoflurane group at T2 and T3 compared to T1 (P < 0.01). In the propofol group, IL-6 and IL-8 were significantly increased at T3 compared to T1. However, there were no significant differences in cytokine concentrations between the isoflurane and propofol groups. CONCLUSION: An inflammatory response occurred earlier in patients who received an inhaled agent compared with an intravenous anesthetic, but no differences in plasma cytokine profiles were evident between isoflurane and propofol anesthesia in patients without comorbidities undergoing minimally invasive surgeries.

Immunophenotypic, immunocytochemistry, ultrastructural, and cytogenetic characterization of mesenchymal stem cells from equine bone marrow.

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium-rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n = 64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies.

Periodontal tissue engineering after tooth replantation.

BACKGROUND: Blood-derived products, platelet-poor plasma (PPP) and platelet-rich plasma (PRP), constitute an approach in the enhancement of tissue healing. PRP has also been used as a scaffold for bone marrow stem cells in tissue engineering. This study evaluates the effect of PPP, calcium chloride-activated PRP (PRP/Ca), calcium chloride- and thrombin-activated PRP (PRP/Thr/Ca), and bone marrow mononuclear cells and PRP/Ca (BMMCs/PRP/Ca) on the healing of replanted dog teeth. METHODS: After 30 minutes of extraction, teeth were replanted with 1) no material (control); 2) PPP; 3) PRP/Ca; 4) PRP/Thr/Ca; or 5) BMMCs/PRP/Ca. Histologic, histomorphometric, and immunohistochemical analysis was assessed 120 days after replantation. Data from histomorphometric analysis were analyzed statistically (analysis of variance, Tukey; P <0.05). Quantitative immunohistochemical analysis was analyzed by Kruskal-Wallis and Dunn post hoc test (P <0.05). RESULTS: Flow cytometry analysis showed 55.98% of CD34(+) and 32.67% of CD90/Thy-1 for BMMCs sample. BMMCs/PRP/Ca presented the largest areas of replacement resorption characterized by osseous ingrowth into cementum (P <0.05), with intense immunomarcation for tartrate-resistant acid phosphatase. The PRP/Ca group also showed areas of replacement resorption with significant immunomarcation for osteopontin. PRP/Thr/Ca presented no replacement resorption. PPP showed areas of inflammatory resorption, with immunomarcation for tartrate-resistant acid phosphatase. CONCLUSIONS: The results suggest that platelets activated with thrombin play an important role in the healing of tissues after tooth replantation. Additional studies are necessary to test other materials, because PRP/Ca did not present an appropriate scaffold for undifferentiated cells in the treatment of avulsed teeth.

Use of in vivo and in vitro systems to select Leishmania amazonensis expressing green fluorescent protein.

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.